Anthrax, a zoonotic disease is caused by gram-positive, sporulating bacteria, Bacillus anthracis. Humans are accidental hosts through food of animal origin, animal products and contamination of the environment with Bacillus anthracis (Brachman P. S., 1970, Aninals. N.Y. Acad. Sci. 174, 577-582). Anthrax is one of the oldest known bacterial diseases and occurs in most parts of the world including India. The major virulent factors of B. anthracis include poly-D-glutamic acid capsule and a three-component anthrax toxin complex. Anthrax toxin (Leppla S. H., 1991, In Source Book of Bacterial protein toxins, pp 277-302.), comprising of protective antigen PA(83 kDa), lethal factor (LF-(90 kDa) and edema factor (EF-(89 kDa) is a major virulent factor of B. anthracis. LF/EF, the catalytic moieties of this complex require PA to enter the cell cytosol. PA in combination with LF (called the lethal toxin), causes death in experimental animals (Smith H. and Keppie J., 1954, Nature, 173, 869-870). PA in combination with EF (called the edema toxin) causes edema in the skin of the experimental animals (Stanley J. L. and Smith H., 1961, J. Gen Microbiol., 26, 49-66). PA is the receptor-binding moiety that facilitates the translocation of the catalytic moieties, LF and EF, into the target cells. After translocation into the cell, LF, a metalloprotease causes cleavage of certain Mitogen so activated protein kinase kinases (MAPKKs) resulting in inactivation of signal transduction pathways (Duesbery N. S., et. al., 1998, Science, 280. 734-737). On the other hand, EF, upon entering the cells, gets activated by calmodulin to cause massive increase in intracellular cAMP levels (Leppla S. H., 1982, Proc. Natl. Acad. Sci. USA., 79, 3162-3166).
The first step of the intoxication process is the binding of PA to the cell surface receptors (Bradley K. A. et al, 2001, Nature, 414 , 225-229). After binding to the receptors on the cell surface, PA gets nicked by cell surface proteases to yield a 63-kDa fragment (Klimpel el R. K., et. al., 1992, Proc. Natl. Acad. Sci. USA., 89, 10277-10281) which oligomerizes and binds to LF/EF (Milne J. C., et. al., 1994, J. Biol. Chem., 269, 20607-20612). Binding of LF/EF is competitive. The whole complex then undergoes receptor-mediated endocytosis. Acidification of the endosonie (Friedlander A. M., 1986, J. Biol. Chem.. 261 , 7123-7126) results in the insertion of the PA-oligomer into the endosomal membrane to form pores (Milne J. C. and Collier R. J., 1993 Mol. Microbiol., 10, 647-653) through which LF/EF are translocated into the cell cytosol.
PA has four domains that are organized primarily into antiparallel-beta sheets with only a few short helices of less than four turns (Petosa C., et. al., 1997, Nature, 385, 833-838). Domain 1 is responsible for binding to LF/EF during the anthrax intoxication process. Domain 2 is dominated by a beta barrel and plays a role in membrane insertion and translocation. Domain 3 is the smallest domain and is important for oligomerization of PA and possibly also in the binding of PA to LF/EF. Domain 4 is the receptor-binding domain.
Crystal structure of LF, determined recently, shows that LF has 4 domains (Pannifer A. D., et al, 2001, Nature, 414, 229-233). Domain 1 is involved in binding to PA. This domain has significant homology to the N-terminal 1-250 residues of EF. In fact, most of the residues in this region are absolutely conserved.
Of all the three toxin proteins, —PA is the most immunogenic and is an essential component of the vaccine against anthrax (Gladstone G. P., 1946, Br. J. Exp. Pathol, 97, 349-418). It has been observed that the protective efficacy of PA is greatly increased if small quantities of LF or EF are incorporated into the vaccine (Pezard et. al., 1995, infect. Immun., 63, 1369-1372). However, this also happens to be the primary reason of toxigenicity and reactogenicity of the vaccines. Anthrax toxin (Leppla S. H., 1991, In Source Book (of Bacterial protein toxins, pp 277-302.), comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF) is a major virulent factor of B. anthracis. 
The currently used anthrax vaccine is derived from a non-capsulated, avirulent strain of the bacterium known as Sterne's strain (Sterne M., 1939, J. Vet. Sci. Anim. Ind, 13, 307-312). In Russia and China, the live spore vaccines based on Sterne strain are used. In UK the vaccine is alum precipitated culture filtrate of the Sterne strain while the US vaccine consists of an alhydrogel-adsorbed cell free culture filtrates of a non-capsulating, non proteolytic derived strain V770 isolated from bovine anthrax (Turnbull P. C. B, 1991, Vaccine, 9, 533-539). All these currently used anthrax vaccines, apart from being crude have undefined composition. They are reactogenic and do not provide protection against all natural strains of B. anthracis. 
U.S. Pat. No. 2,017,606 describes the preparation of anthrax antigen by growing the bacilli with a suitable culture medium, separating the bacilli from the culture medium.
U.S. Pat. No. 2,151,364 describes a method of producing an anthrax vaccine which comprises preparing the suspension of anthrax spores, adding to the suspension a sterile solution containing alum.
RU patent 2,115,433 describes the method of production of anthrax vaccine, which comprises of living spores of non-capsulated strain of B. anthracis and protective antigen of B. anthracis. 
WO patent 000252 describes a method of production of anthrax vaccine using non-toxic protective antigen from B. anthracis for use in inducing immune response, which is protective against anthrax.
The drawbacks in the above-mentioned patents are that all of them use Bacillus anthracis cultures/spores. Bacillus anthracis is an infectious organism and can not be handled without containment facilities. The vaccine prepared is contaminated with other toxic and non-toxic proteins from Bacillus anthracis resulting in a number of side effects and reactogenicity.
These vaccines also have a certain degree of residual virulence for certain species of domesticated and laboratory animals. The Sterne strain is toxigenic and is pathogenic at high doses. As a result it is considered unsafe and unsuitable for human use. This vaccine can cause undesirable side effects including necrosis at the site of inoculation.
Therefore there is a need to develop a second-generation anthrax vaccine which does not have side effects and has a well-defined composition.
The object of the present invention is to render the anthrax toxin non-toxic without affecting its immunogenicity, in order to develop a safe and effective anthrax vaccine.
To achieve said object, the present invention provides a recombinant DNA construct comprising an expression vector and a DNA fragment including genes for wild type Protective Antigen (PA) or wild type Lethal Factor (LF) or wild type Edema Factor (EF)
The present invention also provides a recombinant DNA construct comprising:
an expression vector and a DNA fragment including genes for mutant type Protective Antigen (PA) or mutant type Lethal Factor (LF) or mutant type Edema Factor (EF).
Said vector is a prokaryotic vector such as PQE 30 and said expression vector contains T5 promoter and 6X histidine tag.
The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Phe202.
The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Leu203.
The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Pro205.
The said DNA fragment is the gene for protective antigen with Alanine-substitution at residue Ile207.
The DNA fragment is the gene for protective antigen with Alanine-substitution at residues Pro205, Trp226 and Phe236.
The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Phe552.
The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Ile574.
The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Phe552 and Phe554.
The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Ile562 and Ile574.
The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Leu566 and Ile574.
The DNA fragment is the gene for protective antigen ith Alanine-substitution at residue Phe552 and Phe554, Ile562, Leu566 and Ile574.
The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Phe427.
The DNA fragment is the gene for protective antigen with deletion of residue Asp 425.
The DNA fragment is the gene for protective antigen with deletion of residue Phe 427.
The DNA fragment is the gene for protective antigen With Alanine-substitution at residue Trp346.
The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Leu352.
The DNA fragment is the gene for protective antigen with Alanine-substitution at residue Trp346, Met350 and Leu352.
The DNA fragment is the gene for lethal factor with Alanine-substitution at residue Tyr148.
The DNA fragment is the gene for lethal factor with Alanine-substitution at residue Tyr149.
The DNA fragment is the gene for lethal factor with Alanine-substitution at residue Ile151.
The DNA fragment is the gene for lethal factor with Alanine-substitution at residue Lys153.
The DNA fragment is the gene for lethal factor with Alanine-substitution at residue Asp187.
The DNA fragment is the gene for lethal factor with Alanine-substitution at residue Phe190.
The DNA fragment is the gene for lethal factor with Alanine-substitution at residue Asp 187, Leu188, Leu 189 and Phe 190.
The DNA fragment is the gene for edema factor with Alanine-substitution at residue Tyr137.
The DNA fragment is the gene for edema factor with Alanine-substitution at residue Tyr138.
The DNA fragment is the gene for edema factor with Alanine-substitution at residue Ile140.
The DNA fragment is the gene for edema factor with Alanine-substitution at residue Lys142.
The protein encoded by said DNA fragment is expressed in a prokaryotic host. The said prokaryotic host is an E. coli strain.
A protein expressed by gene DNA fragment is wild type PA wild type LF, wild type EF and their mutagenised variants.
This invention further discloses a method for producing mutagenized anthrax toxin protein comprising:                mutagenizing PA LF & EF genes using different mutagenic primers of the kind as herein defined for PCR reaction;        treating said mutant PCR product along with the native template with a n enzyme to cleave the native template of said PCR product;        transforming said mutant product in E. coli strain;        isolating the recombinant construct from transformed E. coli strain and confirming the desired mutation;        transforming the confirmed mutant construct in appropriate E. coli expression strain to express the mutant protein and        purifying the said expressed mutant protein.        
The purification is carried Out using Ni-NTA chromatography and/or other chromatographic techniques.
The genes are cloned in PQE expression vector containing T5 promoter and 6X histidine tag.
The mutations were affected in the first domain of PA at residues 202, 203, 205. The mutations were affected in the third domain of PA at residues 552, 574 552+554, 562+574, 566+574, 552+554+562+566+574 resulting in mutant proteins that were defective in oligomerization. The mutations were affected in the second domain of PA at residues 425 & 427 of loop 4 of domain 2. These mutations impaired the translocation-ability of PA The mutations were affected in the second domain of PA at residues 346, 352 and 346+350+352 in loop 3 of domain 2 such that PA becomes biologically inactive. The mutations were affected in the 1st domain of LF at residues 148, 149, 151, 153, 187, 190 and 187+188+189+190 impaired the binding of LF to PA. The mutations were affected in the 1st 250 residues of EF.
An anthrax vaccine comprising an anthrax toxin protein is selected from wild type PA or wild type LF or wild type EF.
An anthrax vaccine comprising an anthrax toxin protein selected from mutant type PA or mutant type LF or mutant type EF or a combination thereof.
An anthrax vaccine comprising an anthrax toxin protein selected is a combination of any one selected from wild type PA or wild type LF or wild type EF with any one or more selected from mutant type PA or mutant type LF or mutant type EF.
A pharmaceutical composition comprises an effective amount of an anthrax toxin protein as claimed by the present invention.